Throughout this application various publications are references by Arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
Neuronal cell death is a major feature of a variety of human neurological disorders, including the neurodegenerative diseases (such as Alzheimer's, Parkinson's, Huntington's and amyotrophic lateral sclerosis), stroke and trauma (67). Alzheimer's Disease afflicts about 4 million people in the United States, primarily the elderly. It is characterized by progressive memory loss, disorientation, depression and eventual loss of bodily functions. Amyotrophic lateral sclerosis, afflicts about 30,000 Americans. It begins after age 40 and results in progressive weakness and paralysis. Huntington's Disease, which afflicts an estimated 25,000 patients in the United States, usually begins between the ages of 30 and 50 and includes violent, involuntary movements. Cell death occurs not only as a disease process, but also as a normal aspect of development and of tissue homeostasis (68, 69, 70). Studies of both normal and abnormal cell deaths that occur in organisms as diverse as mammals, insects and nematodes have suggested that many of the distinct primary events that initiate the process of cell death act by triggering one of only a few general mechanisms that cause cells to die (71). If so, an understanding of cell death processes gained from any of these experimental systems might help reveal aspects of the cell death processes that occur in human diseases.
The mechanisms of cell death of one free-living nematode, Caenorhabditis elegans can be studied. In C. elegans, cell death can be observed in living animals at the level of resolution of single, identified cells (11). In addition, C. elegans is easily studied genetically, which not only allows the isolation of mutants with neurodegenerative genetic disorders that might serve as models for human disease, but more generally allows the identification of the genes and proteins that function in cell death. The mechanisms of cell death in wild-type and/or in mutant C. elegans may be similar to those involved in human disease. Furthermore, the genes and gene products involved in C. elegans cell death processes may be sufficiently conserved to allow the identification of corresponding molecules that cause human nerve cell deaths. Recent observations have revealed a striking degree of conservation of gene and protein structure among eukaryotic organisms. For example, many of the genes involved in nervous system development and function in C. elegans or in the fruit fly Drosophila melanogaster have proved to have easily recognized homologs in mammals. Programmed cell death is a prominent feature of C. elegans neural development. For example, the generation of the 816 nongonadal nuclei of the adult hermaphrodite is accompanied by the generation and death of an additional 131 cells (10, 11). About 80% (105/131) of these deaths involve cells that are neural in nature, and 23% (105/463) of all neural cells generated undergo programmed cell death (72).
During the course of C. elegans programmed cell death, a dying cell shrinks, becomes engulfed by a neighboring cell and eventually disappears (10, 11, 73). In the earliest stage of the programmed deaths of cells in the ventral nervous system, which are the cell deaths that have been studied in the most detail, the chromatin forms granular aggregates underlying the nuclear envelop, a cluster of electron-dense particles appears in the center of the nucleus, the nuclear envelop dilates, and both the nucleus and the cytoplasm become more electron dense. Next, the chromatin condenses further (so that very little euchromatin remains visible), the nucleus becomes pycnotic, mitochondria become electron-lucent, and parts of the dying cell split off into membrane-bound fragments. Later, the nuclear membrane becomes highly convoluted, nuclear membrane-bound structures (some containing chromatin-like material) are formed, mitochondria appear distorted (and frequently are found within vacuoles), the cytoplasm appears less granular than before, both internal and plasma membranes display increased whorling, and the cellular outline becomes irregular. Finally, as the dying cell is shrinking, the nuclear membrane breaks down completely, and chromatin-like fragments appear within the cytoplasm. Throughout this process, cytoplasmic extensions from neighboring cells encircle and engulf the dying cell. Genetic studies of C. elegans programmed cell death have defined an 11-gene pathway that functions in all programmed cell deaths (FIG. 8). These genes define three general processes: the death of a viable and potentially functional cell; the engulfment of the dying cell by neighboring cells; and the degradation of residual cellular debris. The one gene identified that functions in this third step is nuc-1 (nuc, nuclease-defective), which encodes or regulates a deoxyribonuclease (DNase) that degrades the DNA in dead cells (74). Seven genes (ced-1, ced-2, ced-5, ced-6, ced-7, ced-8, ced-10) (ced, cell death abnormal) function in the process of phagocytosis of dying cells by their neighbors (74). Mutations that eliminate the nuc-1 DNase activity or that block engulfment do not in general prevent the deaths of cells undergoing programmed cell death, so neither the nuclease nor the process of engulfment is causing these cells to die.
Three genes function in the killing of cells during programmed cell death: ced-, ced-4 and ced-9. Mutations that eliminate the activity of either ced-3 or ced-4 prevent the deaths of all 131 cells that normally die (19). In ced-3 or ced-4 animals, the "undead" cells not only survive, but they also can differentiate and express characteristics of other cells normally present in the animal; different surviving cells differentiate into different cell types. A surviving cell can be sufficiently normal that it is functional: one surviving cell in the animal's pharynx has been shown to acquire characteristics like those normally expressed by its sister, the M4 motor neuron; if the M4 neuron is killed (using a laser microbeam) in a ced-3 mutant animal, the surviving sister of the M4 neuron is capable of replacing it functionally (20). These observations indicate that the genes ced-3 and ced-4 normally act to convert live, potentially functional cells into non-functional cell corpses. In brief, ced-3 and ced-4 cause cells to die.
These "killer genes", ced-3 and ced-4, may act within the dying cells themselves or within other cells that function to cause dying cells to die (75). To determine this, the technique of genetic mosaic analysis was used. Specifically, animals with cells of different genotypes--for example, with some cells wild-type for the ced-3 gene and other cells mutant for the ced-3 gene--were constructed, and it was determined whether the ced genotype of a cell that should die determined whether or not that cell would die in a mosaic animal. The results of these studies revealed that both ced-3 and ced-4 act autonomously, i.e. both of these genes act within dying cells to cause their deaths. These observations indicate that programmed cell death in C. elegans is an active process on the part of dying cells, requiring the functions of gene products that act within the cells that die.
That the two genes known to be required for cells to die during programmed cell death both act within dying cells suggests that the cell death process itself might be cell autonomous. In other words, programmed cell death in C. elegans might be a suicide rather than a murder. A number of other observations are consistent with this hypothesis. For example, many dying cells are smaller than their sisters at the times of their births, suggesting that their fates have already been specified (10, 11). In addition, most dying cells die within an hour of their births, before any overt signs of differentiation (10, 11), which indicates that these cells are unlikely to be dying as a consequence of a failure to compete for targets. Nonetheless, it remains possible that some programmed cell deaths are initiated by cell interactions that activate ced-3 and ced-4 within the cells that die.
The third gene that acts in the killing step of programmed cell death is ced-9. The original ced-9 mutant strain is phenotypically similar to the ced-3 and ced-4 mutants described above: all programmed cell deaths are blocked. However, the ced-9 mutation in this strain is opposite in nature to the ced-3 and ced-4 mutations that have been studied. Specifically, cell death is prevented by mutations that cause a loss of ced-3 and ced-4 gene function or a gain of ced-9 gene function. These observations indicate that whereas ced-3 and ced-4 normally act to cause cells to die, ced-9 might normally act to prevent cells from dying. Further genetic analyses of ced-9 have strengthened the hypothesis that this gene encodes a product that protects cells from programmed cell death.
The studies described above indicate that programmed cell death can be regarded as a cell fate, analogous to any differentiated fate, such as becoming a muscle cell or a dopaminergic neuron. Specifically, all cells that undergo programmed cell death display the same sequence of morphological changes and require the same set of genes, and hence proteins. Thus, the same physiological processes seem to act in all programmed cell deaths in C. elegans. As in the cases of other cell fates, programmed cell death seems likely to involve functions responsible both for determination--the specification of which cells will and which cells will not die--and for differentiation--the expression of the cell death fate itself. The 11 genes discussed above are involved in this latter step, as they carry out programmed cell death. Genes that act in the determinative aspect of programmed cell death would be recognized by their altering patterns of cell death without affecting the machinery necessary for the cell death process. Such genes would include those that could mutate to cause specific cells that normally survive instead to undergo programeed cell death. This phenotype would constitute a degenerative genetic disorder of C. elegans.
One neurodegenerative genetic disorder of this class has been identified in C. elegans. In egl-1 mutants (egl, egg-laying abnormal), the serotonergic HSN motor neurons, which normally innervate the egg-laying musculature and drive egg laying, die (19). No other cells have been found to be abnormal in egl-1 animals. The deaths of the HSNs in egl-1 animals appear morphologically identical to programmed cell deaths, require ced-3 and ced-4 gene function and are blocked by the gain-of-function mutation in ced-9. Thus, mutations in the egl-1 gene cause the highly specific neurodegeneration of the HSN neurons by ectopically activating the program for programmed cell death. It has been proposed (19) that the basis of this phenotype is a sexual transformation in the fate of the hermaphrodite-specific HSN neurons, the homologs of which undergo programmed cell death in males (10). The neurodegenerative phenotype of all egl-1 mutants is dominant.
This invention describes mutations in two other genes, mec-4 (mec, mechanosensory abnormal) (7, 8) and deg-1 (degeneration), which cause neurodegenerative genetic disorders of C. elegans. Unlike egl-1 mutants, in which the genes involved in programmed cell death are ectopically activated, mec-4 and deg-1 mutations cause cells to die independently of the ced genes discussed above. In mec-4 mutants, a specific set of six touch receptor neurons die. In deg-1 mutants, another small set of neurons, including both sensory and interneurons, die. Unlike the cells that die during programmed cell death, the cells that die in these degenerative deaths swell and lyse. The remains of the nucleus and cytoplasmic debris can be seen within a large vacuole many cell diameters in size.